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Woody plant species are often challenging to transform, which complicates gene function studies using molecular biology techniques. To develop an effective gene editing tool for almond (Prunus dulcis, syn. Prunus amygdalus), we established a new method for inducing hairy roots in almond seedlings based on the injection of agrobacterial culture. We generated composite plants comprising wild-type shoots and transgenic hairy roots. This approach was also successful in almond x peach hybrids. The hairy root transformation system was used alongside Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 gene editing to target two transcription factor genes (ETHYLENE RESPONSE FACTOR 74 and GIBBERELLIC ACID INSENSITIVE). We successfully generated hairy roots with knockouts of the target genes and evaluated the efficiency of guide RNAs for gene editing in almond. Our transgenic root method could be a valuable tool for routine gene function studies in almond.Key message: Hairy root induction and composite plant generation were proved to be a suitable system for gene editing using CRISPR/Cas9 in almond, a species highly recalcitrant to transformation. [ABSTRACT FROM AUTHOR]

Background: Light, an essential factor in plant development, exerts a significant impact on both primary and secondary metabolism in plants. Althaea officinalis, commonly known as marshmallow, offers versatile applications through its leaves and roots. With a plethora of identified bioactive compounds and their extensive use in food, health, and supplements, it is widely cultivated globally. This study aimed to demonstrate the definitive positive impact of dark and light irradiation on both primary and secondary metabolite production in A. officinalis hairy roots and to elucidate the light-responsive mechanism through integrated metabolome and transcriptome analysis. Results: When exposed to light, significant changes with a greenish colour shift were observed in 60 metabolites. Multivariate statistical analysis revealed a distinct separation between light- and dark-treated hairy roots, likely attributed to metabolites such as glutamic acid, phenylalanine, catechin hydrate, and chlorophyll. Correspondingly, the pathways significantly impacted included galactose metabolism, alanine, aspartate, and glutamate metabolism, flavone and flavonol biosynthesis, and phenylalanine metabolism. Light-responsive differentially expressed genes associated with pigment and phenylpropanoid biosynthetic pathways were analysed and compared via RNA sequencing. Furthermore, among the light-related transcription factors, including CONSTANS-LIKE and double B-box zinc finger, which are responsible for photomorphogenic modulation, were upregulated. Moreover, light-responsive genes, such as ribulose bisphosphate carboxylase, photosystem II, and chlorophyll A-B binding family protein, were upregulated. Conclusions: These findings emphasise that exposure of A. officinalis hairy root culture to light conditions is a useful method for enhancing most of the primary and secondary metabolites. [ABSTRACT FROM AUTHOR]

Abstract Background Light, an essential factor in plant development, exerts a significant impact on both primary and secondary metabolism in plants. Althaea officinalis, commonly known as marshmallow, offers versatile applications through its leaves and roots. With a plethora of identified bioactive compounds and their extensive use in food, health, and supplements, it is widely cultivated globally. This study aimed to demonstrate the definitive positive impact of dark and light irradiation on both primary and secondary metabolite production in A. officinalis hairy roots and to elucidate the light-responsive mechanism through integrated metabolome and transcriptome analysis. Results When exposed to light, significant changes with a greenish colour shift were observed in 60 metabolites. Multivariate statistical analysis revealed a distinct separation between light- and dark-treated hairy roots, likely attributed to metabolites such as glutamic acid, phenylalanine, catechin hydrate, and chlorophyll. Correspondingly, the pathways significantly impacted included galactose metabolism, alanine, aspartate, and glutamate metabolism, flavone and flavonol biosynthesis, and phenylalanine metabolism. Light-responsive differentially expressed genes associated with pigment and phenylpropanoid biosynthetic pathways were analysed and compared via RNA sequencing. Furthermore, among the light-related transcription factors, including CONSTANS-LIKE and double B-box zinc finger, which are responsible for photomorphogenic modulation, were upregulated. Moreover, light-responsive genes, such as ribulose bisphosphate carboxylase, photosystem II, and chlorophyll A-B binding family protein, were upregulated. Conclusions These findings emphasise that exposure of A. officinalis hairy root culture to light conditions is a useful method for enhancing most of the primary and secondary metabolites. Graphical abstract

Salvia aristata Aucher ex Benth. is considered a valuable endemic medicinal plant in Iran due to its two main bioactive metabolites: phenolic acids and tanshinones. This study established hairy root (HR) cultures from 14-day-old seedlings inoculated with Rhizobium rhizogenes strain ATCC 15834. In addition, the effects of elicitation with Ag+ ions (15 and 25 µM) on growth indices, phenolic acid and tanshinone contents in the HRs, as well as changes in the expression patterns of key genes involved in their biosynthetic pathways were investigated in a time-course experiment. The results showed that exposure of HRs to both Ag+ ions concentrations significantly increased rosmarinic acid (1.34- to 1.43-fold) and salvianolic acid B (1.71- to 1.82-fold) content. Notably, exposure to 25 µM Ag+ ions for seven days resulted in increases of 7.25-, 7.78-, 6.47-, and 3.9-fold in total tanshinones, tanshinone I, tanshinone IIA, and cryptotanshinone levels, respectively, compared to the control groups. The study also revealed a significant increase in the release of tanshinones, especially tanshinone IIA (31.49 ± 0.65 mg L−1) by HRs into the culture medium following Ag+ ions elicitation. Changes in the transcription levels of key genes related to the biosynthetic pathways of phenolic acids (PAL, TAT, and RAS) and tanshinones (CPS and CYP76AH1) were also associated with their contents in the elicited HRs. The findings confirm the effectiveness of elicitation as a strategy to enhance metabolite production in HR cultures of S. aristata, a potent natural source of phenolic acids and tanshinones. Key message: An efficient protocol was established for Salvia aristata hairy root cultures. Additionally, the findings showed that elicitation with Ag+ ions could enhance tanshinone and phenolic acid production in hairy roots. [ABSTRACT FROM AUTHOR]

The Papaver genus is famous for its ability to biosynthesize a wide variety of secondary metabolites, including benzylisoquinoline alkaloids (BIAs) which have been prescribed to treat several health issues, ranging from cough to cancer. Plus, they have been evidenced to be powerful antioxidants scavenging free radical that are synthesized and accumulated when plants are striving to relieve biotic and abiotic stresses. Morphine, codeine, thebaine, noscapine, papaverine, and sanguinarine are the most well-known BIAs. The biosynthesis of BIAs is limited to organized tissues, and because the content of BIAs in these tissues is relatively low, the use of differentiated organ culture, hairy root culture, is drown much more attention. Interestingly, the biosynthetic capacity of the hairy root culture is higher than that of native plants, making them an appropriate platform for in vitro BIA production. One of the most attractive options for improving BIAs accumulation in hairy roots for both modest and massive production is to adopt biotechnological strategies. Regarding the latter, however, the bioreactor-based production of plant bioactive compounds is preceded by optimization of some factors related to machinery and culture medium. The purpose of this review is to supply comprehensive information about current and innovative biotechnological approaches which have been employed or have the potential to be applied for elevating BIA production in the Papaver hairy root culture as well as their importance from the medicinal and defensive perspectives.

Vitex negundo L. is an aromatic, woody, blooming shrub in the Lamiaceae family which can grow into a small tree. Traditionally, V. negundo root has been used to cure diabetes, colic, boils, leprosy, and rheumatism. Keeping the importance of its roots in mind, an attempt has been taken for development of a protocol for efficient hairy root proliferation system. The Agrobacterium rhizogenes strains (MTCC 532 and MTCC 2364) were used for hairy root induction. For A. rhizogenes infection, both in vitro and in vivo leaves as well as internodes were used as explant. In vitro leaves and internodal explants were obtained by the inoculation of matured nodal segments on the optimum medium [MS + 2.0 mg/L N6-Benzylaminopurine (BAP)] with c.a. 91.6% shoot regeneration and an average of 8.1 shoots per explants. In vitro leaf showed best hairy root induction followed by in vitro internode on ½ MS medium augmented with acetosyringone. Highest transformation efficiency was achieved using MTCC 2364 strain, while no transformation was observed in MTCC 532 strain. Different factors affecting transformation including co-cultivation period, infection time and optical density (O.D.) value were standardized. The highest efficacy, 88.8% hairy root induction was observed in in vitro leaves infected by MTCC 2364 for 60 minutes infection time with an O.D. value of 0.29 maintained over a 44-48 hours of co-cultivation period. The prescribed protocol may be used as a reference for development of industrial scale hairy root production for bioactive compound located in root of V. negundo.

Vitex negundo L. is an aromatic, woody, blooming shrub in the Lamiaceae family which can grow into a small tree. Traditionally, V. negundo root has been used to cure diabetes, colic, boils, leprosy, and rheumatism. Keeping the importance of its roots in mind, an attempt has been taken for development of a protocol for efficient hairy root proliferation system. The Agrobacterium rhizogenes strains (MTCC 532 and MTCC 2364) were used for hairy root induction. For A. rhizogenes infection, both in vitro and in vivo leaves as well as internodes were used as explant. In vitro leaves and internodal explants were obtained by the inoculation of matured nodal segments on the optimum medium [MS + 2.0 mg/L N6-Benzylaminopurine (BAP)] with c.a. 91.6% shoot regeneration and an average of 8.1 shoots per explants. In vitro leaf showed best hairy root induction followed by in vitro internode on ½ MS medium augmented with acetosyringone. Highest transformation efficiency was achieved using MTCC 2364 strain, while no transformation was observed in MTCC 532 strain. Different factors affecting transformation including co-cultivation period, infection time and optical density (O.D.) value were standardized. The highest efficacy, 88.8% hairy root induction was observed in in vitro leaves infected by MTCC 2364 for 60 minutes infection time with an O.D. value of 0.29 maintained over a 44-48 hours of co-cultivation period. The prescribed protocol may be used as a reference for development of industrial scale hairy root production for bioactive compound located in root of V. negundo. [ABSTRACT FROM AUTHOR]

This article discusses the optimization of an ex vitro one-step hairy root transformation method in drug- and hemp-type cannabis using the RUBY system. Cannabis is a versatile plant species that has gained attention in medical research and industry due to recent legalization. The concentration of the cannabinoid Δ9‐tetrahydrocannabinol (THC) determines the legal categorization of cannabis as hemp-type or drug-type. The study presents a new method for transforming cannabis roots using the RUBY system, which allows for the visual identification of transformed roots. The results show that the transformation efficiency varied based on the bacterial strain and seed type, with drug-type cannabis exhibiting higher transformation rates. This ex vitro method offers simplicity, speed, and reduced contamination risk, making it suitable for the efficient production of secondary metabolites in cannabis. [Extracted from the article]

Glycyrrhizin, a type of the triterpenoid saponin, is a major active ingredient contained in the roots of the medicinal plant licorice (Glycyrrhiza uralensis, G. glabra and G. inflata), and is used worldwide in diverse applications, such as herbal medicines and sweeteners. The growing demand for licorice threatens wild resources and therefore a sustainable method of supplying glycyrrhizin is required. With the goal of establishing an alternative glycyrrhizin supply method not dependent on wild plants, we attempted to produce glycyrrhizin using hairy root culture. We tried to promote glycyrrhizin production by blocking competing pathways using CRISPR/Cas9-based gene editing. CYP93E3 CYP72A566 double-knockout (KO) and CYP93E3 CYP72A566 CYP716A179 LUS1 quadruple-KO variants were generated, and a substantial amount of glycyrrhizin accumulation was confirmed in both types of hairy root. Furthermore, we evaluated the potential for promoting further glycyrrhizin production by simultaneous CYP93E3 CYP72A566 double-KO and CYP88D6 -overexpression. This strategy resulted in a 3-fold increase (∼1.4 mg/g) in glycyrrhizin accumulation in double-KO/ CYP88D6 -overexpression hairy roots, on average, compared with that of double-KO hairy roots. These findings demonstrate that the combination of blocking competing pathways and overexpression of the biosynthetic gene is important for enhancing glycyrrhizin production in G. uralensis hairy roots. Our findings provide the foundation for sustainable glycyrrhizin production using hairy root culture. Given the widespread use of genome editing technology in hairy roots, this combined with gene knockout and overexpression could be widely applied to the production of valuable substances contained in various plant roots. [ABSTRACT FROM AUTHOR]

Salvia plebeia (Lamiaceae) is a medicinal plant containing diverse bioactive constituents that have biological properties. In this study, we determined the optimal conditions (media and auxin) for the hairy root culture of S. plebeia for the growth and accumulation of phenolic compounds and evaluated its antioxidant activities. Rosmarinic acid and five phenylpropanoids were detected using high-performance liquid chromatography. The hairy roots grown in 1/2 SH medium with 1 mg/L NAA had a high level of rosmarinic acid content. Hairy roots cultured in 1 mg/L NAA had the highest total content of five phenylpropanoids. Compared to wild-type roots grown in the field, hairy roots (NAA 1) expressed similar levels of rosmarinic acid but significantly enhanced phenylpropanoid accumulation. Furthermore, the total phenolic content and total flavonoid content of hairy roots (NAA 1) were 2.22 and 1.73 times higher than those of wild-type roots. In the results of DPPH, ABTS, and reducing power assays, the hairy roots (NAA 1) showed higher free radical scavenging effects and reduction potential than the wild-type roots. These results suggest that S. plebeia hairy roots cultured under optimal conditions, which exhibit enhanced phenolic compound accumulation and antioxidant activity, can potentially be used as sources of antioxidants. [ABSTRACT FROM AUTHOR]

Hypericum perforatum L., commonly known as St. John's wort is an important medicinal plant, belonging to family Hypericaceae. Among all species of Hypericum, H. perforatum is most investigated and exploited. It is sold as one of the world's topmost retailing antidepressants. This plant possesses antibacterial, antiviral, anti-inflammatory, and anticancerous properties. These medicinal properties are attributed to the presence of bioactive compounds, such as hypericins and pseudohypericins (naphthodianthrones), hyperforin and adhyperforin (prenylated acylphloroglucinols), quercetin, rutin, isoquercetin, and catechin (flavonoids), chlorogenic acid, caffeic acid, and tannic acid (phenols) and xanthones. The conventional methods of propagation of H. perforatum are time consuming and field grown plants are exposed to biotic and abiotic challenges which affect its phytochemical constituents. Moreover, these methods are also unable to meet the commercial demand of secondary metabolites. Therefore, in order to meet the growing raw material demand of pharmaceutical industries there is a need to develop effecient in vitro plant regeneration protocols for large scale production of H. perforatum plants and other biotechnological methods (cell, tissue and organ culture, cell suspension culture, hairy root culture, elicitation, etc.) for improvement of target bioactive compounds. The present review provides a comprehensive account of the available information on in vitro plant regeneration and biotechnological approaches used to enhance the secondary metabolite(s) content in H. perforatum during the past years. It also deals with the unexplored areas which might be exploited for drug discovery. [ABSTRACT FROM AUTHOR]

Metabolic engineering improvement of plants will play an essential role in future agriculture, but this largely depends on the establishment of genetic transformation. Stephania tetrandra S. Moore is a traditional Chinese medicine used for rheumatalgia that accumulates benzylisoquinoline alkaloids as its main active ingredients. Wild or farmed plants have remained the main source of these essential medicines, resulting in supply pressure due to the scarcity of wild plant resources and the slow growth rate in cultivation. Here, we constructed Agrobacterium rhizogenes (C58C1)-mediated hairy root culture and a co-transformation system in S. tetrandra to obtain a new source of bisbenzylisoquinoline alkaloids production. We show that the biomass of the hairy roots increased 10-fold, and the content of tetrandrine reached 8.382 ± 0.160 mg/g DW after 50 d of cultivation. In addition, overexpression of (R,S)-norcoclaurine 6-O-methyltransferase (6OMT) gene or treatment of hairy roots with methyl jasmonate (MJ) increased protoberberine alkaloid content. This work provides a method of obtaining hairy roots and a genetic transformation system for S. tetrandra, not only broadening the access to S. tetrandra resources, but also laying a foundation for further elucidation of the biosynthesis of tetrandrine and related alkaloids.

An efficient and reproducible protocol for Agrobacterium rhizogenes-mediated hairy root were efficiently induced from in vitro grown leaf explants of H. enneaspermus. Among the diverse factors analysed, acetosyringone (100 µM), sonication (40 s), and vacuum infiltration (2 min) followed by 2 days co-cultivation period revealed maximal percentage of hairy root induction using A4 strain (29.6%). PCR analysis were confirmed the transgenic status of hairy root by using rol B gene specific primer. Further, the highest amount of fresh weight (17.71 g) and dry weight (2.14 g) was noted at 40-day old A. rhizogenes induced hairy root cultures. Precursor L-tyrosine (100 µM) treated hairy roots produced higher L-Dopa (32.71 mg/g DW) content when compared with control culture. The transformation procedure designed in the current research could be valuable to transferring the gene of interest for secondary metabolism that was introduced into hairy roots of Hybanthus, and it could provide a feasible and sustained product platform for naturally derived, high quality and potential phytochemicals. [ABSTRACT FROM AUTHOR]

Salvia plebeia (Lamiaceae) is a medicinal plant containing diverse bioactive constituents that have biological properties. In this study, we determined the optimal conditions (media and auxin) for the hairy root culture of S. plebeia for the growth and accumulation of phenolic compounds and evaluated its antioxidant activities. Rosmarinic acid and five phenylpropanoids were detected using high-performance liquid chromatography. The hairy roots grown in 1/2 SH medium with 1 mg/L NAA had a high level of rosmarinic acid content. Hairy roots cultured in 1 mg/L NAA had the highest total content of five phenylpropanoids. Compared to wild-type roots grown in the field, hairy roots (NAA 1) expressed similar levels of rosmarinic acid but significantly enhanced phenylpropanoid accumulation. Furthermore, the total phenolic content and total flavonoid content of hairy roots (NAA 1) were 2.22 and 1.73 times higher than those of wild-type roots. In the results of DPPH, ABTS, and reducing power assays, the hairy roots (NAA 1) showed higher free radical scavenging effects and reduction potential than the wild-type roots. These results suggest that S. plebeia hairy roots cultured under optimal conditions, which exhibit enhanced phenolic compound accumulation and antioxidant activity, can potentially be used as sources of antioxidants.

Centella asiatica L., commonly known as Gotu kola, Indian pennywort, and Asiatic pennyworts, is an herbaceous perennial plant that belongs to the family Apiaceae and has long been used in the traditional medicine system. The plant is known to produce a wide range of active metabolites such as triterpenoids including asiatic acid, asiaticoside, brahmoside, and madecassic acid along with other constituents including centellose, centelloside, and madecassoside, etc., which show immense pharmacological activity. Due to its beneficial role in neuroprotection activity, the plant has been considered as a brain tonic. However, limited cultivation, poor seed viability with low germination rate, and overexploitation for decades have led to severe depletion and threatened its wild stocks. The present review aimed to provide up-to-date information on biotechnological tools applied to this endangered medicinal plant for its in vitro propagation, direct or indirect regeneration, synthetic seed production, strategies for secondary metabolite productions including different elicitors. In addition, a proposed mechanism for the biosynthesis of triterpenoids is also discussed. [ABSTRACT FROM AUTHOR]

Rauvolfia serpentina (L). Benth. ex Kurz. (Apocynaceae), commonly known as Sarpagandha or Indian snakeroot, has long been used in the traditional treatment of snakebites, hypertension, and mental illness. The plant is known to produce an array of indole alkaloids such as reserpine, ajmaline, amalicine, etc. which show immense pharmacological and biomedical significance. However, owing to its poor seed viability, lesser germination rate and overexploitation for several decades for its commercially important bioactive constituents, the plant has become endangered in its natural habitat. The present review comprehensively encompasses the various biotechnological tools employed in this endangered Ayurvedic plant for its in vitro propagation, role of plant growth regulators and additives in direct and indirect regeneration, somatic embryogenesis and synthetic seed production, secondary metabolite production in vitro, and assessment of clonal fidelity using molecular markers and genetic transformation. In addition, elicitation and other methods of optimization of its indole-alkaloids are also described herewith. Key points: • Latest literature on in vitro propagation ofRauvolfia serpentina • Biotechnological production and optimization of indole alkaloids • Clonal fidelity and transgenic studies inR. serpentina [ABSTRACT FROM AUTHOR]

In vitro plant propagation systems such as temporary immersion bioreactors (TIBs) are valuable tools that enable production of disease-free plants with improved traits. However, TIB systems can be expensive, difficult to implement, and prone to contamination due to sugar rich propagation media. Using rapidly growing chicory root cultures to expedite design-build-test cycles, we report here an improved, low-cost version of a previously reported Hydrostatically-driven TIB (Hy-TIB) that facilitates economical use of gas mixtures. Bioreactor improvements include decreased material costs, expanded modes of operation, and a horizontal orientation of a plastic film plant growth chambers that increase propagule light exposure. To take advantage of these improvements, we describe here experiments that evaluate the impacts of elevated CO2 on propagation of cacao (Theobroma cacao) secondary embryos and nodal cultures of yam (Dioscorea spp.) during both phototrophic and photomixotrophic growth. Our experiments show that elevated CO2 during plant propagation significantly improved both cacao and yam propagule development and eliminated the need for supplemental sugars in tissue culture growth media. Thus, our improved Hy-TIB shows potential as a simple, low-cost, and scalable propagation platform with cost-effective gas composition control and reduced risk of contamination overgrowth. We provide detailed instructions for assembly of this Hy-TIB design and discuss the implications of its adoption in food-insecure regions of the world. Key message: Elevated CO2 in a temporary immersion bioreactor facilitated removal of sugar from the propagation media, resulting in decreased contamination issues and improved plant root development in agriculturally relevant plant species. [ABSTRACT FROM AUTHOR]

Camptothecin (CPT) is a monoterpenoid-alkaloid, an anticancer compound from plant. Ever since its discovery in 1996 from the bark of Camptotheca acuminata, various researches have been conducted for enhancing its production. CPT has also been reported in several other species belonging to the plant families Icacinaceae, Rubiaceae, Apocynaceae, Nyssaceae, Betulaceae, Violaceae, Meliaceae, and Gelseminaceae. Out of these, Ophiorrhiza sp. (Rubiaceae) is the next possible candidate for sustainable CPT production after C. acuminata and Nothapodytes nimoonia. Various biotechnological-studies have been conducted on Ophiorrhiza sp. for searching the elite species and the most optimal strategies for CPT production. The genus Ophiorrhiza has been used as medicines for antiviral, antifungal, antimalarial, and anticancer activities. Phytochemical analysis has revealed the presence of alkaloids, flavonoids, triterpenes, and CPT from the plant. Because of the presence of CPT and its herbaceous habit, Ophiorrhiza sp. has now become a hot topic in research area. Currently, for mass production of the elite spp., tissue culture techniques have been implemented. In the past decades, several researchers have contributed on the diversity assessment, phytochemical analysis, mass production, and in vitro production of CPT in Ophiorrhiza sp. In this paper, we review the on the biotechnological strategies, optimal culture medium, micropropagation of Ophiorrhiza sp., effect of PGR on shoot formation, rhizogenesis, callus formation, and enhanced production of CPT for commercial use. Key points: • Latest literature on in vitro propagation of Ophiorrhiza sp. • Biotechnological production of camptothecin and related compounds • Optimization, elicitation, and transgenic studies in Ophiorrhiza sp. [ABSTRACT FROM AUTHOR]

Isoflavones (ISF) are potent secondary metabolites, widely distributed among the members of the Fabaceae family. They have a multitude of biological functions in plants, animals, and humans. Most of the functions exhibited by isoflavones are highly beneficial to human beings. Benefits mainly include lowering cardiovascular diseases, reducing menopausal symptoms, and decreasing the risk of hormone-related cancers. Therefore, this potent compound has emerged as a promising possibility for making medicines and has grabbed the pharmaceutical industry's attention. Nowadays, many isoflavone-based products are manufactured commercially as food supplements, and their demand is drastically increasing globally. Numerous in vitro studies have successfully synthesized value-added metabolites that possess a vast number of industrial applications. Production of isoflavones within the plants depends on several biological and environmental conditions. Hence, in vitro cultures provide an alternate source to recover these compounds independent of environmental factors and limited bio-resources. Plant organ cultures could be highly recommended as a reliable platform for synthesizing plant-based pharmaceutically active isoflavones. Moreover, biotechnological approaches, such as elicitation and precursor feeding, can eventually promote the yield and increase these metabolites' production. This review initially outlines information regarding the source, structure, synthesis, and diverse roles of isoflavones. Later, this paper describes different plant organ cultures for the in vitro production of isoflavones and highlights elicitation strategies as well as metabolic engineering work to enhance production. [ABSTRACT FROM AUTHOR]

Plant cell culture systems have become an attractive and sustainable approach to produce high‐value and commercially significant metabolites under controlled conditions. Strategies involving elicitor supplementation into plant cell culture media are employed to mimic natural conditions for increasing the metabolite yield. Studies on nanoparticles (NPs) that have investigated elicitation of specialized metabolism have shown the potential of NPs to be a substitute for biotic elicitors such as phytohormones and microbial extracts. Customizable physicochemical characteristics allow the design of monodispersed‐, stimulus‐responsive‐, and hormone‐carrying‐NPs of precise geometries to enhance their elicitation capabilities based on target metabolite/plant cell culture type. We contextualize advances in NP‐mediated elicitation, especially stimulation of specialized metabolic pathways, the underlying mechanisms, impacts on gene regulation, and NP‐associated cytotoxicity. The novelty of the concept lies in unleashing the potential of designer NPs to enhance yield, harness metabolites, and transform nanoelicitation from exploratory investigations to a commercially viable strategy. [ABSTRACT FROM AUTHOR]

Centella asiatica L., commonly known as Gotu kola, Indian pennywort, and Asiatic pennyworts, is an herbaceous perennial plant that belongs to the family Apiaceae and has long been used in the traditional medicine system. The plant is known to produce a wide range of active metabolites such as triterpenoids including asiatic acid, asiaticoside, brahmoside, and madecassic acid along with other constituents including centellose, centelloside, and madecassoside, etc., which show immense pharmacological activity. Due to its beneficial role in neuroprotection activity, the plant has been considered as a brain tonic. However, limited cultivation, poor seed viability with low germination rate, and overexploitation for decades have led to severe depletion and threatened its wild stocks. The present review aimed to provide up-to-date information on biotechnological tools applied to this endangered medicinal plant for its in vitro propagation, direct or indirect regeneration, synthetic seed production, strategies for secondary metabolite productions including different elicitors. In addition, a proposed mechanism for the biosynthesis of triterpenoids is also discussed.

The peanut plant is one of the most economically important crops around the world. Abiotic stress, such as drought, causes over five hundred million dollars in losses in peanut production per year. Peanuts are known to produce prenylated stilbenoids to counteract biotic stress. However, their role in abiotic stress tolerance has not been elucidated. To address this issue, hairy roots with the capacity to produce prenylated stilbenoids were established. An RNA-interference (RNAi) molecular construct targeting the stilbenoid-specific prenyltransferase AhR4DT-1 was designed and expressed via Agrobacterium rhizogenes -mediated transformation in hairy roots of peanut cultivar Georgia Green. Two transgenic hairy roots with the RNAi molecular construct were established, and the downregulation of AhR4DT-1 was validated using reverse transcriptase quantitative PCR. To determine the efficacy of the RNAi-approach in modifying the levels of prenylated stilbenoids, the hairy roots were co-treated with methyl jasmonate, hydrogen peroxide, cyclodextrin, and magnesium chloride to induce the production of stilbenoids and then the stilbenoids were analyzed in extracts of the culture medium. Highly reduced levels of prenylated stilbenoids were observed in the RNAi hairy roots. Furthermore, the hairy roots were evaluated in a polyethylene glycol (PEG) assay to assess the role of prenylated stilbenoids on water-deficit stress. Upon PEG treatment, stilbenoids were induced and secreted into the culture medium of RNAi and wild-type hairy roots. Additionally, the biomass of the RNAi hairy roots decreased by a higher amount as compared to the wild-type hairy roots suggesting that prenylated stilbenoids might play a role against water-deficit stress. [Display omitted] • Peanut hairy roots with an RNAi construct for a prenyltransferase were made. • Elicitors induced production and secretion of prenylated stilbenoids. • Knockdown of the prenyltransferase reduced prenylated stilbenoid levels. • Water-deficit stress induced prenylated stilbenoid production. • Root biomass was reduced upon water-deficit stress. [ABSTRACT FROM AUTHOR]

The dried root of Astragalus membranaceus is a well-known herbal medicine, and it is useful in treating chronic diseases and weakness, as well as for improving overall health and vitality. Astragalosides, which are root quality indicators of A. membranaceus, are natural triterpenoid saponins that are used in the treatment of diabetes and cardiovascular diseases. Currently, there is an urgent need to improve their production because of their low quantity in plants and the difficulty of chemical synthesis. In this study, yeast extract was added to facilitate elicitation in Agrobacterium-mediated hairy root cultures, thereby enhancing astragaloside production in A. membranaceus. Results showed that yeast extract could stimulate astragaloside content effectively in the hairy roots of A. membranaceus. Moreover, astragaloside accumulation was positively correlated with the upregulation of mevalonate biosynthetic gene expression in the presence of yeast extract. Our study demonstrated that pretreatment with yeast extract (3.65 mM) for 72 h serves as an effective strategy to enhance astragaloside levels in A. membranaceus hairy root cultures. Thus, these optimal conditions can provide valuable information for the improvement of astragaloside industrial production in A. membranaceus. [ABSTRACT FROM AUTHOR]

• Metabolic engineering for curcumin /its glucoside production in Atropa belladonna hairy roots (HR). • First report of heterologous expression of two monocot genes in dicot HR system. • Co-expression of CaUGT2 with DCS & CURS3 genes resulted in curcumin and its glucoside synthesis. • Metabolite productivity and gene-expression profile revealed similar trend. • Bioreactor up-scaling of the transgenic HR clones revealed augmentation in metabolites yield. Curcumin has ignited global interest as an elite drugable molecule, owing to its time-honoured pharmacological activities against diverse human ailments. Limited natural accessibility and poor oral bioavailability caused major hurdles in the curcumin-based drug development process. We report the first successful testimony of curcumin and its glucoside synthesis in Atropa belladonna hairy roots (HR) through metabolic engineering. Re-routing the inherent biosynthetic precursors of the phenylpropanoid pathway of A. belladonna by heterologous expression of key curcumin biosynthetic pathway genes (i.e., Diketide-CoA synthase - DCS and Curcumin synthase - CURS3) and glucosyltransferase gene (CaUGT2) resulted in the production of curcumin and its glucoside in HR clones. Under shake-flask cultivation, the PGD2-HR1clone bearing DCS / CURS3 genes showed the maximum curcumin yield (180.62 ± 4.7 μg/g DW), while the highest content of curcumin monoglucoside (32.63 ± 2.27 μg/g DW) along with curcumin (67.89 ± 2.56 μg/g DW) were noted in the PGD3-HR3 clone co-expressing DCS / CURS3 and CaUGT2 genes. Bioreactor up-scaling showed yield improvements in the PGD2-HR1 (2.3 fold curcumin) and the PGD3-HR3 clone (0.9 and 1.65 folds of curcumin-monoglucoside and curcumin respectively). These findings proved the advantageous use of HR cultures as the production source for curcumin and its glucoside, which remained unexplored so far. [ABSTRACT FROM AUTHOR]